The mammalian intestinal mucosa undergoes a process of continual renewal characterized by active proliferation of stem cells localized near the base of the crypts; progression of these cells up the crypt-villus axis with cessation of proliferation; and subsequent differentiation into one of the four primary cell types (i.e., enterocytes, goblet cells, Paneth cells and enteroendocrine cells). An imbalance in this highly-regimented and orderly process within the crypts is associated with a number of common intestinal pathologies (e.g., colorectal cancer and inflammatory bowel disease [IBD]) which are associated with significant morbidity and mortality. Delineating the molecular factors regulating intestinal proliferation and differentiation is crucial to our understanding of not only normal gut development and adaptation, but also aberrant gut growth. With the support of this grant, we have shown the importance of the PI3K/Akt/mTOR pathway in the regulation of intestinal cell proliferation and differentiation. In addition, we have shown the differential effects of various NFAT isoforms, which integrate and cooperate with the PI3K/mTOR pathway to maintain intestinal homeostasis. As an extension to our published results, our recent findings have demonstrated a novel metabolomic profile associated with intestinal cell differentiation and found that the ketogenic enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) and its product -hydroxybutyrate (HB), an endogenous inhibitor of histone deacetylases (HDACs), are increased with differentiation. In addition, inhibition of mTOR Complex 1 (mTORC1) increases HMGCS2 expression in vitro and in vivo and, moreover, HB induces intestinal cell differentiation. Finally, we have shown that HB increases the expression of the sirtuin proteins that contribute to intestinal cell differentiation; mTOR interacts with and regulates the expressio and deacetylase activity of the sirtuin proteins. Based on our recently published and preliminary findings, we have further refined our central hypothesis to state that intestinal cell proliferatio and differentiation are regulated by PI3K/Akt/mTOR signaling pathway acting on critical downstream proteins (e.g., HMGCS2 and sirtuin proteins); a novel interaction between these pathways may exist that further adds to the intricate, but highly regimented, regulation of intestinal homeostasis. To examine our central hypothesis, we have planned experiments with the following Specific Aims: 1) to delineate mTOR-mediated metabolic reprogramming in intestinal cell proliferation and differentiation; 2) to define the role of HMGCS2/HB in mediatin the effects of mTOR in the intestine; 3) to determine whether sirtuin proteins act cooperatively with HMGCS2/HB to maintain intestinal homeostasis. The studies in the current proposal represent direct extensions of our previous findings, and are designed to define, in a systematic fashion, the molecular mechanisms and signaling events regulating the processes of intestinal proliferation, differentiation, and adaptation.